This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this NINDS-funded project is to identify neuronal substrates for protein phosphatase 5 (PP5). PP5 is a widely expressed Ser/Thr (S/T) phosphatase structurally related to protein phosphatases 1, 2A and calcineurin. PP5 has been implicated in numerous signaling pathways, however little is known concerning its substrates and roles in these pathways and nothing is known concerning its physiologic regulation. We will use a proteomics strategy to determine if PP5 regulates key signaling proteins, to identify novel targets for PP5, and to compare phosphoproteins isolated from cultured neuronal cell lines expressing either wild type PP5 or a mutant form of PP5 with altered sensitivity to the phosphatase inhibitor okadaic acid. Proteins whose phosphorylation status changes as a function of okadaic acid-resistant PP5 activity will be identified and subjected to further analysis generating testable hypotheses for the function and regulation of PP5 in neurons. Phosphopeptides yield low signals during MS analysis because peptide ionization is suppressed by the presence of phosphate groups, and pSer and pThr phosphate groups can be lost during the ionization process. Due to these technical challenges and the need to sample the cellular proteome as broadly as possible, this study requires a sample analysis system with high sensitivity and high throughput capability, experience isolating phosphopeptides and interpreting MS patterns arising from phosphopeptides. Dr. Rossie spent 6 months at PNNL learning sample preparation and processing techniques, IMAC affinity chromatography using on-line capillary LC, and performing pilot studies to explore working strategies for comparing cellular phosphoproteins from matched samples. In her lab, two students are now trained in sample preparation and are generating two types of biological samples;whole cell protein extracts for whole cell phosphoproteomics as described above, and immunopurified complexes of PP5 and binding partners. Samples are being sent to PNNL on an ongoing basis for phosphopeptide purification and analysis utilizing the high sensitivity and high throughput LC/MS systems.